Targeson has developed and commercialized a microbubble contrast agent that is suitable for conjugation to an array of molecular targeting ligands via an avidin/biotin coupling system. The potential immunogenicity and toxicity of the avidin/biotin system, as well as the inconvenience of the ligand coupling, has prompted us to develop a covalently coupled ligand-microbubble platform. We have developed a covalent conjugation system which enables conjugation of ligands to the microbubble surface using thiol-reactive chemistry. This system is non-immunogenic, and requires no user conjugation. We now propose utilizing this microbubble platform to develop a multi-targeted microbubble for imaging tumor angiogenesis. Several angiogenesis-specific endothelial markers have been identified in the context of tumor growth, including VEGFR-2 and avss3 integrin. However, it is known that endothelial cell expression of these and other markers is highly heterogeneous, and the relative expression densities may depend upon the tumor subtype and stage. Recognizing that a molecular imaging probe targeted to a single target may not be useful for detecting angiogenesis unless the target molecule profile is know in advance, we propose a novel multi-targeting strategy. We will construct microbubbles covalently coupled to ligands targeted to both VEGFR-2 and avss3 integrin, with the aim of engineering a robust angiogenesis-targeted agent that can be used to identify angiogenic tumors at diverse stages. We will investigate the adhesion efficiency of dual-targeted microbubbles on recombinant target molecules and endothelial cell substrates in an in vitro flow adhesion assay. We will also evaluate the role of target molecule density and adherent microbubble density on received acoustic signal from dual-targeted microbubbles using an in vitro flow phantom. The multi-targeted microbubble contrast agent developed here will provide a versatile and robust product for ultrasound imaging of a variety of tumor types and facilitate enhanced microbubble retention throughout the course of tumor growth in preclinical research, and has potential for clinical translation. PUBLIC HEALTH RELEVANCE: Ultrasound molecular imaging has emerged as an essential tool in cancer research, as noninvasive imaging of the tumor vasculature enables diagnosis and monitoring of tumor response to therapy. The development of dual-targeted microbubbles constitutes a significant advancement in contrast agent technology, and will facilitate detection and visualization of the regions of tumor growth with greater sensitivity and versatility. Microbubbles pre-targeted to multiple markers of tumor angiogenesis will provide a convenient and user- friendly product for ultrasound imaging of angiogenic tumors in the setting of basic science and drug discovery research, and have tremendous potential for clinical translation. [unreadable] [unreadable] [unreadable]